Characterization of a Streptococcal Endopeptidase with Homology to Human Endothelin-Converting Enzyme

Abstract

A gene encoding an endopeptidase from Streptococcus parasanguis FW213 has been cloned and shown to have high sequence homology to genes encoding mammalian metalloendopeptidases. The gene, designated S. parasanguis pepO, was cloned into the pET28a expression vector, resulting in a fusion of vector sequences encoding a hexahistidine tag at the carboxyl terminus. The recombinant PepO (rPepO) was expressed in Escherichia coli and purified using an Ni 2؉ affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and immunoblot analysis revealed that other oral streptococci contain regions of DNA with homology to pepO and produce a protein with antigenic properties similar to that of FW213 PepO. Enzymatic activity assays indicated that only S. parasanguis species possess the ability to cleave metenkephalin, the natural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that S. parasanguis PepO is a member of the M13 category of metalloendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE-1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that S. parasanguis PepO is more similar to ECE-1 than to NEP.

Streptococcus pyogenes Endopeptidase O Contributes to Evasion from Complement-mediated Bacteriolysis via Binding to Human Complement Factor C1q

“…Endopeptidase O, a zinc metalloendopeptidase, belongs to the M13 peptidase family (24). A number of bacterial species, including Streptococcus dysgalactiae, Streptococcus equi, Streptococcus gallolyticus, Streptococcus parasanguis, Streptococcus thermophilus, Lactococcus lactis, and Lactococcus helveticus, possess its homologue and the active sites of these metalloproteases are all conserved.…”

The Divergently Transcribed Streptococcus parasanguis Virulence-Associated fimA Operon Encoding an Mn 2+ -Responsive Metal Transporter and pepO Encoding a Zinc Metallopeptidase Are Not Coordinately Regulated

“…Streptococcal cultures were grown under specific environmental conditions to early logarithmic growth phase (OD 470 of 0.3 or OD 600 of 0.45) and mid-logarithmic growth phase (OD 470 of 0.6 or OD 600 of 0.9). Whole-cell extracts were prepared as previously described (28) except that cell pellets were resuspended in 20 mM potassium phosphate buffer for immunoblot analyses or 1ϫ reporter lysis buffer (RLB) (Promega Corporation, Madison, Wis.) for luciferase assays. Protein concentrations were determined by using the bicinchoninic acid protein assay reagent kit (Pierce Chemical Company, Rockford, Ill.) with bovine serum albumin as the standard.…”

“…Other members of the LraI family, including PsaA of S. pneumoniae and SloC of Streptococcus mutans, have also been shown to be virulence factors in animal models (4,23). A potential virulence factor in S. parasanguis-induced endocarditis may be the zinc metallopeptidase PepO, which has been shown to be highly homologous in sequence and activity to the vasoconstriction-associated mammalian endothelin-converting enzyme (14,28). The divergently transcribed pepO gene is immediately upstream of the fimA operon.…”

Role of HtrA in Surface Protein Expression and Biofilm Formation by Streptococcus mutans

“…There have been considerable efforts to identify factors of S. mutans and other oral streptococci that are involved in biofilm initiation and development. Surface-associated proteins, such as SpaP and Fap1, function as high-affinity adhesins and are required in the initiation of biofilm (12,23,53). Extracellular glucans, synthesized from sucrose by GTFs, are key players in adhesion interactions and accumulation of mutans streptococci on smooth surfaces (38).…”